Article
Reference
Oligomers of Cyclic Oligochalcogenides for Enhanced Cellular Uptake
MARTINENT, Rémi, et al.
Abstract
Monomeric cyclic oligochalcogenides (COCs) are emerging as attractive transporters to deliver substrates of interest into the cytosol through thiol‐mediated uptake. The objective of this study was to explore COC oligomers. We report a systematic evaluation of monomers, dimers, and trimers of asparagusic, lipoic, and diselenolipoic acid as well as their supramolecular monomers, dimers, trimers, and tetramers. COC dimers were more than twice as active as the monomers on both the covalent and noncovalent levels, whereas COC trimers were not much better than dimers. These trends might suggest that thiol‐mediated uptake of COCs is synergistic over both short and long distances, that is, it involves more than two COCs and more than one membrane protein, although other interpretations cannot be excluded at this level of complexity. These results thus provide attractive perspectives for structural evolution as well as imminent use in practice. Moreover, they validate automated HC‐CAPA as an invaluable method to collect comprehensive data on cytosolic delivery within a reasonable time at a level of confidence that is [...]
MARTINENT, Rémi, et al. Oligomers of Cyclic Oligochalcogenides for Enhanced Cellular Uptake. ChemBioChem, 2021, vol. 22, no. 1, p. 253-259
DOI : 10.1002/cbic.202000630
Available at:
http://archive-ouverte.unige.ch/unige:147189
Disclaimer: layout of this document may differ from the published version.
1 / 1
1
Rémi Martinent, Dongchen Du, Javier López-Andarias, Naomi Sakai and Stefan Matile*
R. Martinent, D. Du, Dr. J. López-Andarias, Dr. N. Sakai and Prof. S. Matile Department of Organic Chemistry
University of Geneva
CH-1211 Geneva, Switzerland E-mail: stefan.matile@unige.ch
Supporting information for this article is given via a link at the end of the document.
Abstract: Monomeric cyclic oligochalcogenides (COCs) are emerging as attractive transporters to deliver substrates of interest into the cytosol through thiol-mediated uptake. The objective of this study was to explore COC oligomers. We report a systematic evaluation of monomers, dimers, and trimers of asparagusic, lipoic, and diselenolipoic acid as well as their supramolecular monomers, dimers, trimers, and tetramers. COC dimers were more than twice more active than the monomers on the covalent and the non-covalent level, while COC trimers were not much better than dimers. These trends might suggest that thiol-mediated uptake of COCs is synergistic over both short and long distances, i.e., involves more than two COCs and more than one membrane protein, although other interpretations cannot be excluded at this level of complexity. These results thus provide attractive perspectives for structural evolution as well as imminent use in practice. Moreover, they validate automated HC-CAPA as an invaluable method to collect comprehensive data on cytosolic delivery within a reasonable time at a level of confidence that is otherwise inconceivable.
2 Introduction
Thiol-mediated uptake is receiving increasing attention as a promising approach to enter into cells.[1-12] Thiol-mediated uptake operates by a dynamic covalent exchange of dichalcogenides and thiols on cell surfaces (Figure 1d). The covalent binding to the cell surface can then be followed by different mechanisms of uptake, including fusion,[1] endocytosis[2] or direct translocation[2-12] into the cytosol, possibly along disulfide tracks[13] and micellar pores[14] (Figure 1e).[3] Cyclic oligochalcogenides (COCs) have been introduced to thiol-mediated uptake to increase the speed and the selectivity of dynamic covalent exchange chemistry on the way into the cytosol.[4] Initial efforts focused on COC ring tension. Relaxed acyclic disulfides and diselenides have a CXXC dihedral angle of 90º to minimize lonepair repulsion and maximize hyperconjugation (Figure 1b).[15] In the 1,2-dithiolane of lipoic acid (LA), the CSSC angle is 35º, in asparagusic acid (AspA) it is 27º (Figure 1c).[4] A gradual increase in ring tension from derivatives of LA to AspA and epidiketodithiopiperazines (CSSC = 0º)[5] was reflected in increasing uptake efficiency. More unorthodox dynamic covalent chalcogenide exchange chemistry was integrated with diselenolipoic acid (DSL)[6] and, so far the most active, benzopolysulfanes (BPS).[7]
Until now, most of COCs explored for uptake were monomeric, also those attached to larger objects such as proteins,[8,9] quantum dots, liposomes and polymersomes.[9] However, linear disulfides, including cell-penetrating poly(disulfide)s (CPDs), become really active only as oligomers or polymers.[2,10-12] Poly(disulfide)s are also routinely used for gene transfection, although emphasis usually is on reductive depolymerization in the cytosol rather than on thiol- mediated uptake.[2] Moreover, the supreme activity of BPS has also been attributed to the in situ evolution of adaptive networks of sulfur-rich oligomers.[7] Mechanistically unrelated oligomer effects are abundant in cellular uptake,[16] arguably best known for oligoarginine CPPs.[17] Considering the high activity of COCs as monomers[4-7] and linear disulfides as
3
oligomers,[10-12] we decided to explore oligomers of COCs. The idea of thiol-mediated uptake with COC oligomers is intriguing considering how little is known about thiol-mediated uptake.
Upon reaction with exofacial thiols, extensive intramolecular exchange could result in either inhibition or activation (Figure 1f). In the context of the working hypothesis of COCs as molecular walkers,[3,8] COC oligomers could be best visualized like animals or insects, and ants arguably coming closest as the transporters of oversized objects.
Figure 1. a) Structure of trifunctional COC monomers and oligomers designed, synthesized and evaluated in this study, with Newman projection of CSSC dihedral angles of b) linear disulfides and c) AspA. d) Thiol-mediated uptake concerns the dynamic covalent exchange of dichalcogenides with exofacial thiols on cell surfaces that e) can be followed by direct translocation, possibly along disulfide tracks and micellar pores, across bilayer membranes. f) This study focuses on thiol-mediated uptake with covalent (n) and supramolecular (m) COC
S S S S S
S S S NH
O O
HN O
O NH HO
O HN
O O
Cl
N NN
HN
HN O N H
S O
n Se Se
O
n R =
AspA
LA
DSL 1 2 3 1 2 3
4 5 6
7 8 9 a)
f)
S S
n’
–S
S S n’
S– S S
S S n’
–S S–
S S
S S n’
d)
n = 1
m = 2 n = 2
e)
R
S 90º
nSσ nSπ R
R S 27º
R b)
c)
NHR
S S O
S S O
4
oligomers, with dimers of dimers (n = m = 2) possibly binding to more than one membrane protein (n’ > 1; m describes the number of peptides that are non-covalently attached to the protein substrate, see below).
Scheme 1. a) Propargylamine, HBTU, TEA, DMF, rt, 15 h, 88%; b) DBU/ CH2Cl2, rt, 20 min, 86%; c) 1. 13, EDCI·HCl, DMAP, CH2Cl2, rt, 1.5 h, 82%; 2. 20% piperidine/DMF, rt, 30 min, 53%; 3. 10, EDCI·HCl, DMAP, CH2Cl2, rt, 15 h, 84%; 4. 20% piperidine/DMF, rt, 30 min, 78%;
5. 13, EDCI·HCl, DMAP, CH2Cl2, rt, 15 h, 91%; 6. 20% piperidine/DMF, rt, 20 min, 84%; 7. 10, EDCI·HCl, DMAP, CH2Cl2, rt, 15 h, 46%; 8. 20% piperidine/DMF, rt, 20 min, 97%; 9. 13, EDCI·HCl, DMAP, CH2Cl2, rt, 6 h, 54%; d) 20% piperidine/DMF, rt, 30 min, 75%; e) 16, EDCI·HCl, DMAP, CH2Cl2, rt, 15 h, 80%; f) 18, CuSO4·5H2O, Na-ascorbate, TBTA, THF/H2O, rt, 1 h, 71%; g) TFA/TIPS/H2O 95/2.5/2.5, rt, 1 h, 99%; h) 21, DIPEA, DMF, rt, 4 h, 26%; use of the activated esters of selenolipoic and lipoic acid instead of 21 yielded trimers 6 (36%) and 9 (48%), respectively (Scheme S3).
d) e)
g)
17 NH
FmocHN
NHBoc O
NH H2N
NHBoc O
OH FmocHN
NHBoc O
a)
b)
c) 10
11
12
O S
O S N O
O
18
NH NNN
HN
NH HN O S
O HN
NHR1 O
O NH R2O
O O O
HN O O NH
HN
NHR1 O
O NH R2O
O HN
NHR1 O
R2O O O
Cl
NH HN
NHBoc O
O tBu-O NH
O O O
HN O O NH
HN
NHBoc O
O NH tBu-O
O HN
NHBoc O
tBu-O O O
Cl
NH HN
NHBoc O
O tBu-O NH
O NHR
HN
NHBoc O
O NH tBu-O
O HN
NHBoc O
tBu-O O O
HO O NHFmoc O
O
OH FmocHN
NHBoc O
h) O
O
HN O
O OH Cl
10 13
16
21 N3
HN
NH HN O S
O
f) 14: R = Fmoc
15: R = H
19: R1 = Boc R2 = tBu
20: R1 = H R2 = H
3
5 Results and Discussion
To explore cellular uptake with COC oligomers systematically, the nine trifunctional peptides 1–
9 were designed, synthesized and studied (Figure 1a). Three of the best developed COCs were selected, i.e., AspA, LA and DSL.[4,5] They were attached to the sidechains of lysines (K) through amide bonds.[18,19] By this operation, the ammonium cations are converted into neutral amides to suppress eventually misleading contributions from CPP-like[14,17] uptake mechanisms.
The resulting K-COCs were alternated with anionic glutamates (E) to minimize passive diffusion across the bilayer membranes and enhance solubility in water. The resulting dyads were repeated n times. The C-terminus of each peptide was equipped with a propargylamine to allow clicking of different substrates, here biotin. Finally, chloroalkanes were attached to the N- terminus to quantify cytosolic delivery by reacting with Halo-tags inside a stable cell line expressing the self-labeling enzyme (HGM cells, see below).
The synthesis of the AspA trimer 3 is outlined in the following as a representative example (Scheme 1). For standard solution phase peptide synthesis, orthogonally protected lysine 10 was condensed first with propargyl amine. With the C-terminal alkyne in place, the N-terminal amine of lysine 11 was selectively deprotected with base. Coupling of the resulting 12 with the orthogonally protected glutamic acid 13 gave the respective dipeptide. The same deprotection and coupling cycles with 10 and 13 were repeated until hexapeptide 14 was reached.
Deprotection of the N-terminus of 14 was followed by coupling of the resulting amine 15 with the chloroalkane 16. The C-terminus of 17 was then “clicked” with azide 18. With the two functional termini in place, all side chains in hexapeptide 19 were deprotected with acid, and coupling of the three amines in 20 with the activated asparagusic acid 21 gave the target molecule 3. All other oligo-COCs were prepared analogously (Schemes S1-S3).
COC oligomers 1–9 were complexed with wild-type streptavidin 22 to probe for protein delivery and to supramolecularly multiply the number of COCs per substrate (Figure 2). The
6
produced complexes 23–34 are described as n.m-COCs, with n COCs per peptide and m peptides per streptavidin. The formal stoichiometry m refers to the equivalents used for preparation; in reality, a statistical mixture of different stoichiometries is generated.[20] Most experiments were done with m = 3, i.e., n.3-COCs, as the maximum number of peptide per streptavidin without the possible presence of unbound peptides.
Figure 2. Binding of COC oligomers 1-9 to streptavidin 22 exemplified with 3. General complex structures are referred to as n.m-COC, with n being the number of COCs per peptide and m the number of peptides per streptavidin, with structure of the streptavidin homotetramer with four bound biotins (yellow).[23]
Most fluorescent quantification methods for cellular uptake lack precise information on localization and functional integrity of the delivered substrate.[1-12,17,21] To improve on this situation, the chloroalkane penetration assay (CAPA) has recently been devisedas a robust and reliable method for the quantification of cytosolic delivery (Figure 3).[22] CAPA operates with
S S
22
LA DSL AspA
m = 3
23 24 25
26 27 28
29 30 31 1
2 3 n
1 2 3 4 m
32 33 30 34 DSL n = 2
=
m = 3
3 3.0-AspA
25 3.3-AspA
=
HN O
O NH HO
O NH HN O
O HO NH
O
O O
HN O
O HO NH
O HN
O O
Cl
N NN
HN
NH HN O S
O
n = 3 NH
O
S S NH O
S S NH O
7
HGM cells, which are stably modified HeLa Kyoto cells expressing a HaloTag-GFP construct on the outer part of the mitochondrial membrane, pointing into the cytosol (Figure 3a). Cell- penetrating chloroalkane-containing molecules react with a carboxylate in the catalytic site of the HaloTags in the cytosol to form a covalent ester bond (Figure 3b). All unreacted HaloTags are then labeled with chloroalkylated rhodamines 35 that freely diffuse into the cells (Figure 3c).
In CAPA, fluorescence intensity is thus inversely proportional to cytosolic uptake efficiency.
This fluorescence is usually measured by flow cytometry.[22] Recently, we have combined CAPA with high-content high-throughput (HCHT) fluorescence imaging to further maximize accuracy.[8]
HC-CAPA has been explicitly assessed and described in detail.[23] In brief, it operates based on HT microscopy and automated data analysis with highly precise quantification methods.
Moreover, the same HCHT screen can inform on cytotoxicity by detecting the number of abnormal cells with early signs of apoptosis or damage mitochondrial networks during automated data analysis.
For HC-CAPA, varying concentrations of protein-transporting AspA oligomers 23–25 were incubated with HGM cells. After four hours, the cells were washed and incubated with rhodamine 35, followed by Hoechst 33342, and then mitochondrial fluorescence intensities were recorded. Hill analysis provided the CP50, the concentration needed to reach a 50% reduction of fluorescence intensity, i.e., half-maximal cell penetration. The concentrations used in the following are those of the streptavidin tetramer.
Streptavidin tetramers loaded with three AspA monomers, that is 1.3-AspA 23, gave a CP50
= 7.4 ± 0.5 μM (Figure 4a, b, Table 1, entry 1). With three AspA dimers in 2.3-AspA 24, the activity almost tripled to CP50 = 2.6 ± 0.2 μM. With three AspA trimers in 3.3-AspA 25, the activity further increased a little to CP50 = 2.0 ± 0.2 μM.
8
Figure 3. a) CAPA for quantitative detection of cytosolic delivery operates with stable cells with HaloTag and GFP expressed on their mitochondria (HGM). b) Chloroalkylated transporters that reach the cytosol will covalently react with the HaloTag. c) Chloroalkylated cell-permeable rhodamine 35 is then added to label all unreacted HaloTags.
35 c)
exofacial thiols
HaloTag GFP mitochondria
b)
HS
HS NH
O O Cl
O
O N+
N COOH
=
a)
–S
25 3.3-AspA
2.2 µM
9
Figure 4. Dependence on oligomer length: HC-CAPA dose-response curves for a) 1.3-AspA 23 (q), 2.3-AspA 24 (n) and 3.3-AspA 23 (l), c) 1.3-DSL 29 (q), 2.3-DSL 30 (n) and 3.3-DSL 31 (l), and d) 1.3-LA 26 (q), 2.3-LA 27 (n) and 3.3-LA 28 (l), with b) CP50 values of 23–25.
The same trends were found with DSL in place of AspA as the COC. The monomers 1.3- DSL 29 gave CP50 = 17 ± 1 μM and failed to reach full inhibition at 20 µM (Figure 4c, Table 1, entry 7). The activity with dimers 2.3-DSL 30 more than doubled to CP50 = 7.1 ± 0.6 μM, whereas trimers 3.3-DSL 31 did not much improve to CP50 = 6.0 ± 0.3 µM.
However, the trends were most pronounced with LA as COC. The monomers 1.3-LA 26 were essentially inactive up to 20 µM (Figure 4d, Table 1, entry 4). The CP50 = 40 ± 30 μM represents a speculative extrapolation. In sharp contrast, the activity of dimers 2.3-LA 27
0.1 1 10
0.0 0.5 1.0
Concentration (μM)
Normalized intensity
1.0
0.5
0.1
I(rel)
1 10
c (μM) a)
0.0
0.1 1 10
0.0 0.5 1.0
Concentration (μM)
Normalized intensity
0.1 1 10
0.0 0.5 1.0
Concentration (μM)
Normalized intensity
1.0
0.5
0.1
I(rel)
1 10
c (μM) 23 1.3-AspA
7.4 µM
25 3.3-AspA
2.0 µM 24
2.3-AspA 2.6 µM
c)
0.0
1.0
0.5
0.1
I(rel)
1 10
c (μM) d)
0.0 b)
Asp A-mo
nomer Asp
A-d ime
r
Asp A-trime 0 r
2 4 6 8 10
Compounds CP50 (µM)
24 25 23 10
CP50 (μM) 8 6 4 2 0
10
jumped to CP50 = 2.3 ± 0.2 µM. This corresponded to an about 18-fold increase in activity from monomer to dimer. Moving on to trimers 3.3-LA 28, activities slightly decreased to CP50 = 4.3
± 0.2, presumably because of limited solubility.
Cross-comparison of HC-CAPA curves was revealing on the monomer level. AspA was confirmed as the best, with CP50 = 7.4 ± 0.5 µM for 1.3-AspA 23 (Figure 5a, c). Consistent with lower ring tension, i.e., reactivity,[3-8] LA was the weakest, with 1.3-LA 26 essentially inactive up to 20 µM. On the dimer and trimer level, activities of different COCs were more similar, with all COCs ending up CP50 = 2–6 µM, AspA remaining the best (Figure 5b, d).
Figure 5. Dependence on the COCs: HC-CAPA dose-response curves for a) 1.3-LA 26 (l), 1.3-DSL 29 (n) and 1.3-AspA 23 (q), and b) 3.3-DSL 31 (◆), 3.3-LA 28 (n) and 3.3-AspA 25 (l), with CP50 values of c) monomers compared to d) trimers.
Beyond covalent COC oligomers, contributions from supramolecular oligomers were explored next, using DSL dimer 8 as an example. Complex formation with just one equivalent,
AspA13 DSL13 LA13 0
10 20 30
CP50 (μM) 10
0 20 30
25 31 28
CP50 (μM)
0.1 1 10
0.0 0.5 1.0
Concentration (µM)
Normalized intensity
1.0
0.5
0.1
I(rel)
1 10
c (μM) b)
0.0 23
1.3-AspA 7.4 µM
26 1.3-LA 41 µM 29
1.3-DSL 17 µM
0.1 1 10
0.0 0.5 1.0
Concentration (μM)
Normalized intensity
AspA13 DSL13 LA13 0
10 20 30
CP50 (µM)
10 0 20 30 1.0
0.5
0.1
I(rel)
1 10
c (μM) a)
0.0
23 29 26
c) d)
11
i.e., 2.1-DSL 32, gave with CP50 = 19 ± 4 μM relatively poor activity, just achieved at the 20 µM limit (Figure 6, Table 1, entry 10). The transition from one to two peptides per protein, i.e., 2.2- DSL 33, more than doubled the activity to CP50 = 7.6 ± 0.6 μM. With three and four ligands, activities did not further change much. The gradually continuing underadditivity until 2.4-DSL 34 nicely confirmed[8] the quantitative formation of tetramer 34 from one equivalent of protein 22 and four equivalents of peptide 8, and thus, the absence of free 2.0-DSL 8.
Like with covalent oligomers, the activity of supramolecular dimers was thus overadditive, while the activity of higher oligomers was underadditive. In the streptavidin homotetramer, two biotin-binding sites are close to each other but on opposite sides of the other two (Figures 2, 6).
Two COCs bound to streptavidin can thus be cis or trans, but an excess of trans is generally preferred to minimize steric and charge repulsion.[20] This would imply one COC oligomer, at least a dimer, on each face of the protein as active structure, which in turn would suggest the presence of long-range synergism. Such synergism over long distances could imply that exofacial thiols and disulfide tracks[3,8,13] of more than one membrane protein (including homodimers, e.g., transferrin receptor,[18] etc) contribute to the thiol-mediated uptake of COCs (Figure 1f). Combined with short-range synergism in covalent dimers, this conclusion could support dimers of dimers as lead structure with overall more than three COCs reacting during thiol-mediated uptake.
It is very important to highlight that it is not possible to exclude alternative interpretations at this level of complexity. Secondary interactions of various nature could conceivably contribute to, or even account for, the overadditive performance of dimers of dimers, rather than multiple synergistic dynamic covalent chalcogenide exchanges. The same call for caution applies for the general, somewhat puzzling underadditivity with higher oligomers, both covalent and supramolecular. It might well support dimers of dimers as lead structure, with additional COCs contributing little to function. It also might originate from systematic limitations of so far unknown
12
nature at these very high activities. Decreasing solubility with increasing oligomer length, for instance, is a common problem. However, such effects should not be general. They should affect LA and particularly DSL more than AspA. The observed CP50 values were not at the lowest limit of HC-CAPA.[23] Passive diffusion by small molecules has been detected down to CP50 ~ 20 nM,[24] and longer incubation times drove the CP50 of COC oligomers also into the nanomolar range (vide infra).
Table 1: Summary of uptake data for COC oligomers.[a]
Entry Complex [b] n[c] m[d] t [h] [e] CP50 [µM] [f]
1 23 1.3-AspA 1 3 4 7.4 ± 0.5
2 24 2.3-AspA 2 3 4 2.6 ± 0.2
3 25 3.3-AspA 3 3 4 2.0 ± 0.2
4 26 1.3-LA 1 3 4 40 ± 30
5 27 2.3-LA 2 3 4 2.3 ± 0.2
6 28 3.3-LA 3 3 4 4.3 ± 0.2
7 29 1.3-DSL 1 3 4 17 ± 1
8 30 2.3-DSL 2 3 4 7.1 ± 0.6
9 31 3.3-DSL 3 3 4 6.0 ± 0.3
10 32 2.1-DSL 2 1 4 19 ± 4
11 33 2.2-DSL 2 2 4 7.6 ± 0.6
12 34 2.4-DSL 2 4 4 6.6 ± 0.5
13 24 2.3-AspA 2 3 2 5.0 ± 0.3
13
14 30 2.3-DSL 2 3 0.3 >100
15 30 2.3-DSL 2 3 1 37 ± 12
16 30 2.3-DSL 2 3 2 25 ± 7
17 30 2.3-DSL 2 3 10 3.0 ± 0.4
18 27 2.3-LA 2 3 0.3 14 ± 1
19 27 2.3-LA 2 3 1 8.4 ± 0.6
20 27 2.3-LA 2 3 2 5.7 ± 0.9
21 27 2.3-LA 2 3 10 0.8 ± 0.1
[a] 96-well plates with HGM cells were incubated for time t[e] with serial dilutions of COCs[b-d] in Leibovitz’s medium at 37 °C. Then, cells were washed, incubated with 35 for 15 min, washed, incubated with Hoechst 33342 for 15 min, washed and imaged over three channels (377/477, 475/536, 531/593 nm). [b] See Figures 1a and 2 for structure and notations. [c] Number of COCs per peptide (Figure 1a). [d] Number of peptides per protein (Figure 2). [e] Incubation time. [f] Concentration of half-maximal cell penetration, from HC-CAPA curves, Figures 4-7.
The activities found for COC oligomers are significant. For instance, they are in the range of the CP50 = 3.1 ± 0.5 µM reported for the Tat peptide, the original CPP.[21] The effective activity of the most active COC trimer 3.3-AspA 25 (CP50 = 2.0 ± 0.2 μM) is much higher, considering that they transport a large protein and add up to nine extra negative charges, and that in general uptake decreases with negative charges and substrate size.[9,11] Moreover, the activity of COC oligomers was comparable to the ones previously reported for the best cell-penetrating streptavidin (CPS) with COC monomers covalently linked through triazole linkage on the surface of the protein.[8] In the CPS series, eight AspA monomers covalently attached to the protein were with CP50 = 8 ± 1 μM four times less active than 3.3-AspA 25 equipped with three trimers (CP50 = 2.0 ± 0.2 μM).[8] Similarly, CPS with four covalently attached DSL were nearly cell-
14
impermeable (CP50 = 34 ± 6 μM),[8] whereas 2.2-DSL 33 bearing two dimers rather than four monomers showed good activity (CP50 = 7.6 ± 0.6 μM).
Figure 6. Dependence on supramolecular oligomers: a) HC-CAPA dose-response curves for 2.1-DSL 32 (cyan), 2.2-DSL 33 (light blue), 2.3-DSL 30 (blue) and 2.4-DSL 34 (dark blue), with b) CP50 values of 2.m-DSLs 30, 32–34.
The CP50 of 2.3-AspA 24 depended clearly on incubation time. Recorded after two instead of four hours of incubation, it increased to CP50 = 5.0 ± 0.3 μM (Figure 7a, Table 1, entry 13).
Time dependence of HC-CAPA was thus investigated more systematically with 2.3-DSL 30 and 2.3-LA 27. For each time point, HC-CAPA curves were recorded (Figure 7c, d). The dependence of the obtained CP50 on time gave the respective t50 and CP50MAX (27: CP50MAX = 1.1 ± 0.7 µM, 30: CP50MAX = 1.9 ± 3.4 µM) (Figure 7b). With t50 = 58 min, the more active 2.3- LA 27 entered the cytosol only slightly faster than the less active 2.3-DSL 30 with t50 = 78 min.
Slightly faster uptake of LA compared to DSL oligomers is contrary to their reactivity with
34 2.4-DSL
6.6 µM 33
2.2-DSL 7.6 µM 32
2.1-DSL 18.6 µM
1 eq. 2 eq. 3 eq. 4 eq.
0 5 10 15 20 25
Compounds CP50 (µM)
0.1 1 10
0.0 0.5 1.0
Concentration (µM)
Normalized intensity CP50 (μM)
10
0 20 1.0
0.5
0.1
I(rel)
1 10
c (μM) a)
0.0
32 33 34
b)
30 30
2.3-DSL 7.1 µM
15
thiolates.[6] Uptake with LA and DSL monomers was in agreement with this intrinsic reactivity, 1.3-LA 26 being 2.4-times less active than 1.3-DSL 29 (Figure 5a, Table 1, entry 4, 7). Inversion of this trend with dimers, here trimers of dimers 2.3-LA 27 outperforming 2.3-DSL 30 in efficiency (up to 4.4-times), confirmed the previous conclusion that the less active LA benefits most from synergistic oligomer effects on both the molecular and the supramolecular level.
After ten hours of incubation, 2.3-LA 27 reached a submicromolar final CP50 = 0.8 ± 0.1 μM, i.e. entered the nanomolar range of activities (Figure 7d, Table 1, entry 21). As already mentioned above, this activity for COC-mediated cytosolic protein delivery is quite remarkable compared to the uptake of the much smaller HIV-Tat, an undecapeptide with CP50 = 3.1 ± 0.5 μM, and also the best CPS with CP50 = 2.1 ± 0.3 μM.[8,22]
Figure 7. Dependence on time: HC-CAPA dose response curves of a) 2.3-AspA 24 after 2 h (q) and 4 h (n) of incubation, c) 2.3-DSL 30 after 20 min (p), 1 h (yellow l), 2 h (n), 4 h (red l) and 10 h (brown l) of incubation and d) 2.3-LA 27 after 20 min (dotted red l), 1 h (dashed red l), 2 h (solid red l), 4 h (brown l), and 10 h (pale brown l) of incubation (increasing activities with time), with b) kinetics for 2.3-DSL 30 (l) and 2.3-LA 27 (n).
20 220 420 620
0 20 40
CP50 (µM)
20 10
20 CP50 (μM)
220 420
t (min) b)
0
0.1 1 10
0.0 0.5 1.0
Concentration (µM)
Normalized intensity
0.1 1 10
0.0 0.5 1.0
Concentration (µM)
Normalized intensity
0.1 1 10
0.0 0.5 1.0
Concentration (μM)
Normalized intensity
1.0
0.5
0.1
I(rel)
1 10
c (μM) a)
0.0
1.0
0.5
0.1
I(rel)
1 10
c (μM) c)
0.0
1.0
0.5
0.1
I(rel)
1 10
c (μM) d)
0.0
620 30
40
16 Conclusion
The objective of this study was to explore cytosolic delivery with COC oligomers of different nature systematically. A trifunctional peptide scaffold is introduced to produce a comparable set of monomers, dimers and trimers of asparagusic acid (AspA), lipoic acid (LA) and selenolipoic acid (DSL) together with tags for non-covalent oligomerization and quantitative detection of function in the cytosol. Such comprehensive sets of data are rare to find in studies on cellular uptake. It highlights the practical importance of automated high-throughput HC- CAPA to access rich, robust and statistically relevant data collections within reasonable time.[8,22] Manual execution of the same experiments would not be possible at this level because it would be too inaccurate, and simply take too long.
We found that moving from COC monomers to dimers, activities increase for both covalent and supramolecular systems in an overadditive manner. Moving from dimers to trimers and beyond, activities further increase, but the increase is underadditive. These trends could imply dimers of dimers as a new lead structure. Moreover, they could suggest that cytosolic delivery by thiol-mediated uptake is not limited to simple dynamic covalent exchange of one COC with an exofacial thiol. The apparent synergy within covalent oligomers could have mechanistic implications for the steps following this first covalent attachment to the cell surface. Further, dynamic covalent exchange on the way into the cell could support the so far purely speculative hypothesis of molecular walkers moving along disulfide tracks in membrane proteins[13] (Figure 1d-f). The additional synergy between covalent oligomers attached at opposite sides of large substrates, here proteins, could further imply that more than one membrane protein is involved in thiol-mediated uptake by direct translocation, including possible homodimers. Although alternative conclusions can never be excluded at this level of complexity, the emerging impression that more than one COC and more than one membrane protein account for thiol-
17
mediated uptake is in agreement with previous hypotheses[3,5,8] as well as ongoing extensive inhibitor screens.
Dimers of dimers as a possible new lead structure should not be overestimated. The underadditivity observed with higher oligomers could originate from effects unrelated to cellular uptake, such as solubility. This question is currently addressed with the dependence of COC trimers on peptide length, sequence, stereochemistry, charge, and so on. These synthetic efforts are meaningful because the activities obtained with COC oligomers are interesting. This promise in practice can be difficult to judge hands-off. Here, our optimism is based on preliminary use of COC trimers carrying noncovalently interfaced substrates for targeted delivery and controlled release,[8] which works so far very well.
Acknowledgements
We thank D. Moreau and S. Vossio for help with HC-CAPA recording and analysis, J. A. Kritzer (Tufts University) and T. R. Ward (University of Basel) for providing materials, the ACCESS automated microscopy, high-content high-throughput screening facility and the NMR, MS and Bioimaging platforms for services, and the University of Geneva, the NCCR Chemical Biology, the NCCR Molecular Systems Engineering and the Swiss NSF for financial support.
Keywords: Cyclic Oligochalcogenides • Oligomers • Cellular Uptake • Thiol-Mediated Uptake
[1] a) E. P. Feener, W. C. Shen, H. J. Ryser, J. Biol. Chem. 1990, 265, 18780–18785; b) H. J.- P. Ryser, R. Flückiger, Drug Discov. Today 2005, 10, 1085–1094.
[2] a) S. Ulrich, Acc. Chem. Res. 2019, 52, 510–519; b) X. Meng, T. Li, Y. Zhao, C. Wu, ACS Chem. Biol. 2018, 13, 3078–3086; c) J. Zhou, Z. Shao, J. Liu, Q. Duan, X. Wang, J. Li, H.
Yang, ACS Appl. Bio. Mater. 2020, DOI 10.1021/acsabm.9b01131; d) S. Du, S. S. Liew, L.
Li, S. Q. Yao, J. Am. Chem. Soc. 2018, 140, 15986–15996; e) D. Oupický, J. Li, Macromol.
18
Biosci. 2014, 14, 908–922; f) Y. Zhang, Y. Qi, S. Ulrich, M. Barboiu, O. Ramström, Mater.
Chem. Front. 2020, 4, 489–506; g) R. Bej, P. Dey, S. Ghosh, Soft Matter 2019, 16, 11–26;
h) A. Kichler, J. S. Remy, O. Boussif, B. Frisch, C. Boeckler, J. P. Behr, F. Schuber, Biochem.
Biophys. Res. Commun. 1995, 209, 444–450; i) S. Aubry, F. Burlina, E. Dupont, D.
Delaroche, A. Joliot, S. Lavielle, G. Chassaing, S. Sagan, FASEB J. 2009, 23, 2956–2967;
j) A. G. Torres, M. J. Gait, Trends Biotechnol. 2012, 30, 185–190; k) L. Brülisauer, N.
Kathriner, M. Prenrecaj, M. A. Gauthier, J.-C. Leroux, Angew. Chem. Int. Ed. 2012, 51, 12454–12458; l) M. Balakirev, G. Schoehn, J. Chroboczek, Chem. Biol. 2000, 7, 813–819;
m) C. R. Drake, A. Aissaoui, O. Argyros, M. Thanou, J. H. G. Steinke, A. D. Miller, J. Control.
Release 2013, 171, 81–90.
[3] Q. Laurent, N. Sakai, S. Matile, Helv. Chim. Acta 2019, 102, e1800209.
[4] G. Gasparini, G. Sargsyan, E.-K. Bang, N. Sakai, S. Matile, Angew. Chem. Int. Ed. 2015, 54, 7328–7331.
[5] L. Zong, E. Bartolami, D. Abegg, A. Adibekian, N. Sakai, S. Matile, ACS Cent. Sci. 2017, 3, 449–453.
[6] N. Chuard, A. I. Poblador-Bahamonde, L. Zong, E. Bartolami, J. Hildebrandt, W. Weigand, N. Sakai, S. Matile, Chem. Sci. 2018, 9, 1860–1866.
[7] Y. Cheng, L. Zong, J. López-Andarias, E. Bartolami, Y. Okamoto, T. R. Ward, N. Sakai, S.
Matile, Angew. Chem. Int. Ed. 2019, 58, 9522–9526.
[8] J. López-Andarias, J. Saarbach, D. Moreau, Y. Cheng, E. Derivery, Q. Laurent, M.
González-Gaitán, N. Winssinger, N. Sakai, S. Matile, J. Am. Chem. Soc. 2020, 142, 4784–
4792.
[9] a) E. Bartolami, D. Basagiannis, L. Zong, R. Martinent, Y. Okamoto, Q. Laurent, T. R. Ward, M. Gonzalez-Gaitan, N. Sakai, S. Matile, Chem. Eur. J. 2019, 25, 4047–4051; b) N. Chuard,
19
G. Gasparini, D. Moreau, S. Lörcher, C. Palivan, W. Meier, N. Sakai, S. Matile, Angew.
Chem. Int. Ed. 2017, 56, 2947–2950.
[10] Z. Shu, I. Tanaka, A. Ota, D. Fushihara, N. Abe, S. Kawaguchi, K. Nakamoto, F. Tomoike, S. Tada, Y. Ito, Y. Kimura, H. Abe, Angew. Chem. Int. Ed. 2019, 58, 6611–6615.
[11] a) E.-K. Bang, G. Gasparini, G. Molinard, A. Roux, N. Sakai, S. Matile, J. Am. Chem. Soc.
2013, 135, 2088–2091; b) G. Gasparini, E.-K. Bang, G. Molinard, D. V. Tulumello, S. Ward, S. O. Kelley, A. Roux, N. Sakai, S. Matile, J. Am. Chem. Soc. 2014, 136, 6069–6074; c) P.
Yuan, X. Mao, X. Wu, S. S. Liew, L. Li, S. Q. Yao, Angew. Chem. Int. Ed. 2019, 58, 7657–
7661; d) J. Lu, H. Wang, Z. Tian, Y. Hou, H. Lu, J. Am. Chem. Soc. 2020, 142, 1217–1221;
e) J. Zhou, L. Sun, L. Wang, Y. Liu, J. Li, J. Li, J. Li, H. Yang, Angew. Chem. Int. Ed. 2019, 58, 5236–5240; f) X. Zhang, R. M. Waymouth, J. Am. Chem. Soc. 2017, 139, 3822–3833;
g) Y. Liu, Y. Jia, Q. Wu, J. S. Moore, J. Am. Chem. Soc. 2019, 141, 17075–17080; h) E.
Derivery, E. Bartolami, S. Matile, M. Gonzalez-Gaitan, J. Am. Chem. Soc. 2017, 139, 10172–10175; i) P. K. Hashim, K. Okuro, S. Sasaki, Y. Hoashi, T. Aida, J. Am. Chem. Soc.
2015, 137, 15608–15611; j) Q. Zhang, Y. X. Deng, H. X. Luo, C. Y. Shi, G. M. Geise, B. L.
Feringa, H. Tian, D. H. Qu, J. Am. Chem. Soc. 2019, 141, 12804–12814.
[12] N. Chuard, G. Gasparini, A. Roux, N. Sakai, S. Matile, Org. Biomol. Chem. 2015, 13, 64–
67.
[13] a) C. Alvadia, N. K. Lim, V. Clerico Mosina, G. T. Oostergetel, R. Dutzler, C. Paulino, eLife 2019, 8, e44365; b) J. Wu, Z. Yan, Z. Li, X. Qian, S. Lu, M. Dong, Q. Zhou, N. Yan, Nature 2016, 537, 191–196; c) S. Jing, I. S. Trowbridge, EMBO J. 1987, 6, 327–331; d) H. A. Khan, B. Mutus, Front. Chem. 2014, 2, 70; e) J. Tombling, C. K. Wang, D. J. Craik, Angew. Chem.
Int. Ed. 2020, 59, 11218–11232; f) L. Zhao, F. Bai, F. Chen, M. Guo, L. Gan, H. Zhang, J.
Fang, Chem. Commun. 2020, 56, 2857–2860; g) Z. Yang, D. H. Kang, H. Lee, J. Shin, W.
Yan, B. Rathore, H.-R. Kim, S. J. Kim, H. Singh, L. Liu, J. Qu, C. Kang, J. S. Kim,
20
Bioconjugate Chem. 2018, 29, 144 6–1453; h) Law, B. K.; Castellano, R. K. PCT Int. Appl.
2019, WO 2019241644 Al.
[14]a) N. Chuard, K. Fujisawa, P. Morelli, J. Saarbach, N. Winssinger, P. Metrangolo, G. Resnati, N. Sakai, S. Matile, J. Am. Chem. Soc. 2016, 138, 11264–11271; b) G. Gasparini, E. K.
Bang, J. Montenegro, S. Matile, Chem. Commun. 2015, 51, 10389–10402.
[15] a) A. D. Clauss, S. F. Nelsen, M. Ayoub, J. W. Moore, C. R. Landis, F. Weinhold, Chem.
Educ. Res. Pract. 2014, 15, 417–434; b) P. C. Hiberty, B. Braïda, Angew. Chem. Int. Ed.
2018, 57, 5994–6002. R. Singh, G. M. Whitesides, J. Am. Chem. Soc. 1990, 112, 6304–
6309.
[16] a) K. C. Tjandra, P. Thordarson, Bioconjugate Chem. 2019, 30, 503–514; b) C. Gehin, J.
Montenegro, E.-K. Bang, A. Cajaraville, S. Takayama, H. Hirose, S. Futaki, S. Matile, H.
Riezman, J. Am. Chem. Soc. 2013, 135, 9295–9298; c) G. A. Eggimann, E. Blattes, S.
Buschor, R. Biswas, S. M. Kammer, T. Darbre, J.-L. Reymond, Chem. Commun. 2014, 50, 7254–7257; d) K. Petkau-Milroy, M. H. Sonntag, A. H. A. M. van Onzen, L. Brunsveld, J. Am.
Chem. Soc. 2012, 134, 8086–8089; e) J. Haensler, F. C. Szoka, Bioconjugate Chem. 1993, 4, 372–379; f) R. Gillies, J. M. J. Fréchet, Drug Discov. Today 2005, 10, 35–43; g) R. Esfand, D. A. Tomalia, Drug Discov. Today 2001, 6, 427–436; h) R. Haag, Angew. Chem. Int. Ed.
2004, 43, 278–282.
[17] a) I. Lostalé-Seijo, I. Louzao, M. Juanes, J. Montenegro, Chem. Sci. 2017, 8, 7923–7931;
b) J. Hatai, C. Schmuck, Acc. Chem. Res. 2019, 52, 1709–1720; c) T. Murayama, T. Masuda, S. Afonin, K. Kawano, T. Takatani-Nakase, H. Ida, Y. Takahashi, T. Fukuma, A. S. Ulrich, S. Futaki, Angew. Chem. Int. Ed. 2017, 56, 7644–7647; d) S. A. Bode, R. Wallbrecher, R.
Brock, J. C. M. van Hest, D. W. P. M. Löwik, Chem. Commun. 2014, 50, 415–417; e) C. J.
McKinlay, R. M. Waymouth, P. A. Wender, J. Am. Chem. Soc. 2016, 138, 3510–3517; f) M.
Juanes, I. Lostalé-Seijo, J. R. Granja, J. Montenegro, J. Chem. Eur. J. 2018, 24, 10689–
21
10698; g) S. L. Kuan, D. Y. W. Ng, Y. Wu, C. Förtsch, H. Barth, M. Doroshenko, K. Koynov, C. Meier, T. Weil, J. Am. Chem. Soc. 2013, 135, 17254–17257; h) A. F. L. Schneider, A. L.
D. Wallabregue, L. Franz, Hackenberger, C. P. R. Bioconjugate Chem. 2019, 30, 400–404;
i) M. Whitney, E. N. Savariar, B. Friedman, R. Levin, J. L. Crisp, H. L. Glasgow, R. Lefkowitz, S. R. Adams, P. Steinbach, N. Nashi, Q. T. Nguyen, R. Y. Tsien, Angew. Chem. Int. Ed.
2013, 52, 325–30; j) D. M. Copolovici, K. Langel, E. Eriste, U. Langel, ACS Nano 2014, 8, 1972–1994; k) A. Walrant, A. Bauzá, C. Girardet, I. D. Alves, S. Lecomte, F. Illien, S. Cardon, N. Chaianantakul, M. Pallerla, F. Burlina, A. Frontera, S. Sagan, Biochim. Biophys. Acta 2020, 1862, 183098; l) H. D. Herce, A. E. Garcia, M. C. Cardoso, J. Am. Chem. Soc. 2014, 136, 17459–17467; m) F. Sgolastra, B. M. Deronde, J. M. Sarapas, A. Som, G. N. Tew, Acc.
Chem. Res. 2013, 46, 2977–2987; n) D. Schmidt, Q. Jiang, R. Mackinnon, Nature 2006, 444, 775–779; o) N. Sakai, S. Matile, J. Am. Chem. Soc. 2003, 125, 14348–14356.
[18] D. Abegg, G. Gasparini, D. G. Hoch, A. Shuster, E. Bartolami, S. Matile, A. Adibekian, J.
Am. Chem. Soc. 2017, 139, 231–238.
[19] A. Tirla, P. Rivera-Fuentes, Biochemistry 2019, 58, 1184–1187.
[20] a) M. Fairhead, D. Krndija, E. D. Lowe, M. Howarth, J. Mol. Biol. 2014, 426, 199–214; b) X.
Sun, D. Montiel, H. Li, H. Yang, Bioconjugate Chem. 2014, 25, 1375–1380; c) A. Loosli, U.
E. Rusbandi, J. Gradinaru, K. Bernauer, C. W. Schlaepfer, M. Meyer, S. Mazurek, M. Novic, T. R. Ward, Inorg. Chem. 2006, 45, 660–668; d) A. Goujon, K. Straková, N. Sakai, S. Matile, Chem. Sci. 2019, 10, 310–319; e) T. Heinisch, T. R. Ward, Acc. Chem. Res. 2016, 49, 1711–
1721; f) C. L. Schreiber, B. D. Smith, Nat. Rev. Chem. 2019, 3, 393–400.
[21] a) A. Méndez-Ardoy, I. Lostalé-Seijo, J. Montenegro, ChemBioChem 2019, 20, 488–498; b) K. Deprey, L. Becker, J. Kritzer, A. Plückthun, Bioconjugate Chem. 2019, 30, 1006–1027; c) A. Lacroix, E. Vengut-Climent, D. de Rochambeau, H. F. Sleiman, ACS Cent. Sci. 2019, 5, 882−891.
22
[22] a) L. Peraro, K. L. Deprey, M. K. Moser, Z. Zou, H. L. Ball, B. Levine, J. A. Kritzer, J. Am.
Chem. Soc. 2018, 140, 11360–11369; b) K. Deprey, J. A. Kritzer, Methods Enzymol. 2020, 641, 277–309.
[23] R. Martinent, J. López-Andarias, D. Moreau, Y. Cheng, N. Sakai, S. Matile, Beilstein J. Org.
Chem. 2020, 16, 2007–2016.
[24] K. Straková, J. López-Andarias, N. Jiménez-Rojo, J. E. Chambers, S. J. Marciniak, H.
Riezman, N. Sakai, S. Matile, ACS Cent. Sci. 2020, 6, 1376–1385.
23
